Hi Francesco,

Is there any possibility that it´s a virus?

Hi Konstantin,

I don't know if they could be virus, people gave to us the culture already fixed

I found something in this site https://synapseweb.clm.utexas.edu/2302, maybe the structure marked by the arrow could me similar to those I found in my samples.

Thank you very much Konstantin

Hi Francesco,

I also thought about lysosomes but these organelles are always very dense by EM. Another possibility is peroxisosme but based on EM atlases I can not say it neither. Since microglia is a part of immune cells I thought it might be some special structures of cells functionality and that's why I asked about a virus. 

As another possibility is to check whether it's a secretory vesicles. Look for instance, this link https://www.uni-mainz.de/FB/Medizin/Anatomie/workshop/EM/EMSekretE.html

Hi Konstantin,

I thought too about some secretory vesicles. There are many possibilities.

Thank you very much!!!

Dear Francesco,

it could also help to inform us a bit about the processing proper (culture conditions, fixation (duration, 1st, 2nd, OsO4?) , dehydration,embedding, resin type, section thickness, staining your ultrathins). Having ha d a short look into the pdf regarding the structures in question I also don't have a clue for now....but think that these structures are too big for virus particles....(size / area of textures almost as large as mitochondria. Part of their content eluted by processing?) Thanking in advance, best, WM

Hi Wolgang,

I will ask about culture conditions and I will write about the embedding.

Thank you very much and happy Easter!!!

@Francesco: E anche voi Buona Pasqua!

looking forward to your further blog, warm regards, Wolfgang

Hi,

here the informations about cultures and embedding.

The Microglial primary cultured cells have been fixed in 2.5% glutaraldehyde in sodium cacodylate buffer 0.1M 1h at 4° C, post fixed in osmium tetroxide 1% + potassium ferrocyanide 1% 1h at 4° C, dehydrated in EtOh 25%, 50%, 75%, 96% 1X10 min at 4° C, EtOh 100% 3x15 min, EtOH:Epon 1:1 1h at 37° C, then Epon ON at 37° C and at 60° C for two days. The sections were about 80 nm thickness and stained with uranyl acetate and lead citrate.

Thank you in advance

Hi Francesco,

Do you have by chance a possibility to do a High-Pressure Freezing instead of Chemical preparation of samples ? 

Best,

I would like! But I don't have the device.

Caro Francesco, thank you for informing us about the processing of the cultured cells. I have re-viewed the images you provided (BTW-excellent EM-micrographs) and would like to reason about the textures in question:  IMHO it is

NO virus or virus-like structure.

It is / these structures are  - at least- remnants of  'blown-up' "organelles" with double membrane, some "cristae-like" protrusions from those in the direction of the center which in a certain manner is lacking "stained" substrate (cf. esp. image A: ) which could be eluted "lipid" or lipid-like matter/debris. It is known that cells in culture - and especially the / their mitochondria  undergo degenerating changes, especially when the are stressed anyhow (we do not know for now about such stress factors whether such are induced by their treatment, their culture conditions or [*]the final fixation and processing into resin)

[*]:  as of your protocol, to me processing at 4°C is unnecessary...I am certain that applying fixative etc.etc. at Room temperature will not induce "more" textural alterations. The starting of dehydration in 25% EtOH followed by 50% seems at least to be unconconventional - since it has been reported that starting with 30% EtOH CAN induce textural alterations (like "maceration") for matrix or texture substrata  if these tissular textures aren't "fixed-conserved and stabilized completely"..... Also - it might be, that osmication + ferrocyanide for delicate cell structures (I anticipate as +/- a monolayer culture) over 1 hour [even if it is at 4°C] might be too long [as compared to the aldehyde fixation for 1 hour too). "Lipid" or "lipid-like material" storage in stressed mitochondria of cultured cells has been reported.... and could be the equivalent image you see after our processing into resin.

The only thing I could / would like to recommend is to check for such lipidic material in your "vacuolar structures" (which I consider to be altered mitiochondria) would be:

i) after osmication (and washing step in respective buffer) start with 50% EtOH (if you want to start with 4°C  -- but let warm up to RT and apply all other steps at RT),

then ii) proceed with dehydration in 2 parallel batches as follows:

iia)  transfer half of cells on culture plate / well  from 50% EtOH into plain 70% EtOH

iib)   transfer the other half of cells on culture plate / well from 50% EtOH into 1% PPD (= para-phenylene diamine) in 70% EtOH for 10-15 min, afterwards wash at least twice in plain 70%EtOH (at least until you can't see further "brown PPD-bleeding" from the culture 

then proceed    iii a&b [= both batches) with 80, 90, 96, 100% EtOH 5-10 min at RT (you see: you will not have the problems with hygroscopic and adsorptive properties of higher alcohols at 4°C), infiltrate and embed as you do that routinely.

IF the "empty vacuoles" in vivo contain lipid/lipidic substrate (neutral lipids, + eventually associated with glycolipids etc., cf. e.g.: *) )  you will find that in the PPD-stabilized specimens because of (better) retention of certain substrata which have been not fixed 120% by your fixation procedere     [*)  cf:  https://phys.libretexts.org/LibreTexts/University_of_California_Davis/UCD%3A_Biophysics_241/Glycolipids ] you'll find "+/- full vacuoles" with stainable "matrix".

Sorry for lengthiness....If you would like to see a very short pamphlet why I used PPD in ALL of my former professional "diagnostic" biopsy specimens, please link to: 

https://www.researchgate.net/publication/200468964_Electron_Microscopy_as_an_important_diagnostic_tool_in_Neuronal_Ceroid-Lipofuscinosis_NCL_Batten%27s_Disease

 and

https://www.researchgate.net/publication/200468954_PRO%27s_for_a_multipleRepeated_use_and_Safe_Disposal_with_Recovery_of_OsmiumtetroxideOsO4_solutions_in_Routine_Electron_Microscopy_EM. 

EDIT: Note added 2017-04-20,12:20 MEST:  As I found some orthographic errors and - as well - lacking words / letters in this my reply No. 11 .... which may lead to some confusion....  I honestly shall create Re# 14 which is the corrected form of this reply (hoping that I found all mistakes I made yesterday). 

Conference Paper PRO's for a multiple/Repeated use and Safe Disposal with Rec...

Conference Paper Electron Microscopy as an important diagnostic tool in Neuro...

Thank you very much Wolfgang for your clear suggestions!!!

Dear Francesco, you're welcome.

As I found some orthographic errors and - as well - lacking words/letters in my reply No. 11, I honestly shall create  Re# 15 which is the corrected form (hoping that I found all mistakes I made yesterday). 

Thank you again!

EDIT: Note 2017-04-20,12:20 MEST:

As I found some (too many) orthographic errors and - as well - lacking words / letters in my previous reply No. 11 ....  which may lead to some confusion....  I honestly post  the corrected reply as Re# 15 (hoping that I found all mistakes I made yesterday).

"

 Caro Francesco, thank you for informing us about the processing of the cultured cells. I have re-viewed the images you provided (BTW-excellent EM-micrographs) and would like to reason about the textures in question:  IMHO it is

- NO virus or virus-like structure.

- It is / these structures are  - at least- remnants of  'blown-up' "organelles" with double membrane, some "cristae-like" protrusions from the latter in the direction to the center / core of structure, which in a certain manner is lacking "stained" substrate (cf. esp. image A: ). This perhaps could be attributed to or interpreted as poorly retained or  ‚eluted‘ "lipid" or lipid-like matter / debris.

It is known that cells in culture - and especially the / their mitochondria  undergo degenerating changes, especially when they are stressed anyhow (we do not know for now about such stress factors, e. g. whether such are induced by their treatment, their culture conditions or [*]the final fixation and processing into resin)[*]:  

[*] as of your protocol, to me processing at 4°C is unnecessary...   I am certain that -  in your morphological approach  - applying fixative and other solutions at room temperature will not induce "more" textural alteration(s)  than we have to expect at all after chemical fixation. The starting of dehydration in 25% EtOH followed by 50% ethanol  to me seems at least to be unconconventional = individual   - since it has been reported that starting with 30% EtOH CAN induce textural alterations (like "maceration") for matrix or texture / substrata if these tissular textures aren't "fixed - conserved and stabilized completely".....  Also - it might be, that osmication + ferrocyanide for delicate cell structures (I anticipate as +/- a monolayer culture) over 1 hour [even if it is at 4°C] might be too long [as compared to the aldehyde fixation for 1 hour too).

"Lipid" or "lipid-like material" storage in stressed mitochondria of cultured cells has been reported.... and therefore the vacuolar structures could resemble the ‚equivalent ultrastructural image‘ you see after your processing into resin.

 The only thing I could / would like to recommend is to check for such lipidic material in your "vacuolar structures" (which I consider to be altered mitochondria) which can be done as follows:

i) after osmication (and washing step in respective buffer) start with 50% EtOH (if you initially started with 4°C during fixation,  then at least after osmication let solutions warm up to RT and apply all other processing steps at RT),

then

ii) proceed with dehydration in 2 parallel batches as follows:

iia)   transfer half of cells on culture plate / well  from 50% EtOH into plain 70% EtOH

iib)   transfer the other half of cells on culture plate / well from 50% EtOH into 1% PPD (= para-phenylene diamine) in 70% EtOH for 10-15 min,

afterwards wash at least twice in plain 70%EtOH (at least until you can't see further "brown PPD-bleeding" from the fixed cells adhering to the culture plate / wells.

Then proceed further:  

iiia&b [= both batches) with 80%, 90%, 96%, 100% 100%, 100% EtOH 5-10 min at RT (you see: you will not have the problems with hygroscopic and adsorptive properties of higher alcohols at 4°C), infiltrate and embed as you do that routinely.

IF the "empty vacuoles" in vivo contained lipid / lipidic substrate (e. g. neutral lipids, + eventually associated / filled with glycolipids etc.  [cf. e. g.: **)] )  you will find "+/- full vacuoles" with stainable "matrix" in the PPD-stabilized specimens. This because certain substrata -  which may have been not fixed „100%“ by your fixation procedere – may have been (better) retained within the cells..

[**)  cf:  https://phys.libretexts.org/LibreTexts/University_of_California_Davis/UCD%3A_Biophysics_241/Glycolipids ]

Sorry for lengthiness.... If you would like to see two very short pamphlets [why I used PPD in ALL of my former professional "diagnostic" biopsy specimens]  please link to (A) cf. appearance of vacuoles in Fig.2): 

A) https://www.researchgate.net/publication/200468964_Electron_Microscopy_as_an_important_diagnostic_tool_in_Neuronal_Ceroid-Lipofuscinosis_NCL_Batten%27s_Disease 

 and (B) cf. appearance of vacuoles in Fig.1 compared to Fig.2) in:

B) https://www.researchgate.net/publication/200468954_PRO%27s_for_a_multipleRepeated_use_and_Safe_Disposal_with_Recovery_of_OsmiumtetroxideOsO4_solutions_in_Routine_Electron_Microscopy_EM"

Naturally I could provide the "specific" literature and references related to the expanded processing method -  if there is need for. Best wishes and good luck, WM.

Conference Paper PRO's for a multiple/Repeated use and Safe Disposal with Rec...

Conference Paper Electron Microscopy as an important diagnostic tool in Neuro...